Uterine contractility is affected by a variety of hormones. Substances such as beta-adrenergic agents which promote relaxation also increase uterine cAMP levels. Agents such as prostaglandins and oxytocin, which cause contractions, have been shown in certain instances to antagonize relaxant-generated cAMP levels and may also elevate cGMP levels. The pregnant rat uterus shows decreased spontaneous contractions and responsiveness to oxytocin relative to nonpregnant uterus, measured as antagonism of isoproterenol effects on contractility and cAMP levels. Recent evidence from biochemical and physiological experiments suggested that a rapidly acting substance acts in addition to progesterone to achieve this effect. Uterine relaxing factor (URF) and relaxin, both of ovarian origin, have been variously described as identical and distinguishable, homogenous and resolvable into closely related polypeptides. The objectives of the present study are to explore the mechanism of action of purified URF as it relates to the role of cyclic nucleotides in the regulation of uterine motility in the rat and to further delineate the biochemical interrelationships between hormones known to interact in the uterus. It is proposed to (1) study the effects of existing relaxin preparations on uterine contractility and cAMP levels; (2) study the effect of relaxin on cAMP levels alone and in combination with oxytocin; (3) purify URF from pregnant bovine ovaries and compare its biological and physical properties with existing URF and relaxin preparations; (4) assess the role of relaxin in promoting the decreased responsiveness of the pregnant uterus; (5) study simultaneous effects of purified URF on uterine contractility and biochemical parameters and attempt to elucidate the details of the mechanisms involved; and (6) attempt to find URF receptors in the uterus.